1. Field of the Invention
The present invention relates to the field of molecular biology. The method of the invention is useful to increase both the specificity and the fidelity of PCR or other assays that employ polymerases.
2. Description of the Prior Art
Detecting and identifying variations in DNA sequences among individuals and species has provided insights into evolutionary relationships, inherited disorders, acquired disorders and others aspects of molecular genetic and medicine.
These variations may involve different lengths of DNA, from several nucleotides down to just a single one. The detection of single nucleotide polymorphisms (SNPs) is a challenging task aimed to provide new developments in the field of Molecular Biology.
The analysis of sequence variation has traditionally been performed by restriction fragment length polymorphism (RFLP) in a Southern blot format, or more recently, by digesting PCR products. The RFLP analyses are based on a change in the restriction fragment length as a result of a change in the sequence. Nowadays most techniques rely on the differential annealing of allele-specific oligonucleotides to a template. Some of these techniques are allele-specific oligonucleotide hybridization (ASO), reverse dot blot, competitive oligonucleotide priming (COP), primer extension sequence test (PEST), nucleic acid depolymerization (READIT®), and amplification refractory mutation system (ARMS), also known as allele-specific PCR (ASP), PCR amplification of specific alleles (PASA) and allele-specific amplification (ASA).
A key aspect of the methods that are based upon oligonucleotide base-pairing is that the allele-specific oligonucleotide must anneal only to the homologous sequence to prevent misleading results. However, this is not always the case with the methods where 3′ mismatches are used to identify the different alleles. Newton et al. (Nucleic Acids Res. 17: 2503, 1898) and Kwok, et al. (Nucleic Acids Res. 18: 999, 1990) report that a 3′ terminal mismatch on the PCR primer produced variable results, making it necessary to add a 3′ terminal mismatch accompanied by a second mismatch within the last four nucleotides of the primer. The arbitrariness in the addition of extra mismatches near the 3′ end on individual primers in every particular instance limits the general application of the technique in a simple and universal fashion. Also, because of the lower selectivity due to the formation of false DNA synthesis products when using single 3′ mismatch primers, the informative power of the gene variation analyses and gene mutation analyses is limited. The formation of false DNA synthesis products can lead to false findings, as a result of which concerning risks arise for the patient and for biomedical research in general.
The U.S. Pat. No. 6,403,313, teaches methods to detect specific hybridization between single-stranded probes and non-denatured double-stranded targets to form triplex by an intercalating agent, thus obviating the need to denature the target. This method can be used to determine the number of mismatched pairs in a hybridization complex, and to map genomes.
Bodmer et. al. (WO 01/75155), teach methods that can distinguish between specific and non-specific amplification products, for example adding to the post-amplification products an amount of small molecules sufficient to increase the pH of the sample products, wherein the pH is 11-14 and then assaying the post-amplification sample product in order to detect and/or quantify any double-stranded nucleic acid present. The method is useful for detecting and/or quantifying a specific double-stranded nucleic acid amplification product in a nucleic amplification reaction post-amplification sample, as in ARMS-PCR methods for SNP typing.
U.S. Pat. No. 5,639,611 discloses an allele specific PCR reaction with two primers (mutant and normal alleles), which one of the primers is complementary to the first allele, but which primer forms a mismatch with the second allele at the 3′ end of the primer, employing a DNA polymerase wherein the first allele is specifically amplified but little or no amplification the second allele occurs.
U.S. patent application Ser. No. 10/009,761 disclose a method for detecting a single nucleotide polymorphism in a target by isothermal nucleic acid amplification, hybridizing a detector primer to the target wherein the detector primer comprises a diagnostic nucleotide for the single nucleotide polymorphism about one to four nucleotides from 3′ terminal nucleotide of the detector primer, which is complementary to the target sequence, amplifying the target, determining an efficiency of detector primer extension and detecting de presence or absence of the single nucleotide polymorphism based on the efficiency of detector primer extension. This application disclosed the ARMS method.
U.S. Pat. No. 6,312,894 disclosed an hybridization and mismatch discrimination using oligonucleotides conjugated to minor grooves binders. The minor grooves binders is a molecule having a molecular weight of approximately 150 to 2,000 Daltons as 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate that binds in a non-intercalating manner into de minor groove of a double-stranded nucleic acid.
On the other hand, the use of high fidelity DNA polymerases in the polymerase chain reaction (PCR) is essential for reducing errors in the amplification of products that are intended for cloning, sequencing and expression. Several DNA Polymerases with 3′-5′ exonucleaseproofreading activity (Pfu, Vent, Deep Vent) are currently used for such high fidelity applications. However, Taq DNA polymerase, which is the most used enzyme in routine PCR, does not have a proofreading activity and shows a fairly low fidelity (Tindall, K. R. and Kunkel, T. A. (1988) Biochemistry 28:6008). Some improvements on the fidelity of Taq DNA polymerase have been reported using pH 5-6 or equimolar concentrations of MgCl2 and dNTPs (Eckert, K. A. and Kunkel, T. A. (1990) Nucl. Acids. Res. 18:3739).
It would be therefore convenient to have a method to increase the selectivity of gene variation analyses and, by suppressing the formation of false positives, to prevent wrong diagnoses and erroneous findings. The goal of the present invention is to increase the selectivity in specific nucleic acids sequence analyses adding an intercalating agent to the conventional reactions medium, reducing the efficiency of primer extension by polymerase when the 3′ end of a primer does not hybridize perfectly with the target sequence. The addition of the intercalating agent avoids the need to place a second mismatch in the sequence of the detector primer which is not directed to detection or identification of the allele of interest. Since the priming selectivity repeats in every following nucleotide addition to the growing chain, it is also another embodiment of the present invention to increase the fidelity of the amplification of nucleic acid fragments, in particular, those which will be used in cloning, expression and all other applications where overall low error rates in nucleic acid chains may be desired.